Korean J Physiol Pharmacol.  2014 Feb;18(1):79-86. 10.4196/kjpp.2014.18.1.79.

Phosphorylation of Akt Mediates Anti-Inflammatory Activity of 1-p-Coumaroyl beta-D-Glucoside Against Lipopolysaccharide-Induced Inflammation in RAW264.7 Cells

• Vo VA
• Lee JW
• Kim JY
• Park JH
• Lee HJ
• Kim SS
• Kwon YS
• Chun W

• KMID: 2285495
• DOI: http://doi.org/10.4196/kjpp.2014.18.1.79

Abstract

Hydroxycinnamic acids have been reported to possess numerous pharmacological activities such as antioxidant, anti-inflammatory, and anti-tumor properties. However, the biological activity of 1-p-coumaroyl beta-D-glucoside (CG), a glucose ester derivative of p-coumaric acid, has not been clearly examined. The objective of this study is to elucidate the anti-inflammatory action of CG in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells. In the present study, CG significantly suppressed LPS-induced excessive production of pro-inflammatory mediators such as nitric oxide (NO) and PGE2 and the protein expression of iNOS and COX-2. CG also inhibited LPS-induced secretion of pro-inflammatory cytokines, IL-1beta and TNF-alpha. In addition, CG significantly suppressed LPS-induced degradation of IkappaB. To elucidate the underlying mechanism by which CG exerts its anti-inflammatory action, involvement of various signaling pathways were examined. CG exhibited significantly increased Akt phosphorylation in a concentration-dependent manner, although MAPKs such as Erk, JNK, and p38 appeared not to be involved. Furthermore, inhibition of Akt/PI3K signaling pathway with wortmannin significantly, albeit not completely, abolished CG-induced Akt phosphorylation and anti-inflammatory actions. Taken together, the present study demonstrates that Akt signaling pathway might play a major role in CG-mediated anti-inflammatory activity in LPS-stimulated RAW264.7 macrophage cells.

Keyword

1-p-coumaroyl beta-D-glucoside (CG); Akt; COX-2; iNOS; Lipopolysaccharide; NF-kappaB; RAW264.7 cells

MeSH Terms

Coumaric Acids
Cytokines
Dinoprostone
Glucose
Inflammation*
Macrophages
NF-kappa B
Nitric Oxide
Phosphorylation*
Tumor Necrosis Factor-alpha
Coumaric Acids
Cytokines
Dinoprostone
Glucose
NF-kappa B
Nitric Oxide
Tumor Necrosis Factor-alpha

Figure

• 

  Fig. 1 Chemical structure of 1-p-coumaryol β-D-glucoside (CG).

• 

  Fig. 2 CG attenuated LPS-induced NO and PGE2 production in RAW264.7 macrophage cells. RAW264.7 cells were pretreated with various indicated concentrations of CG for 1 hr, then incubated with LPS (200 ng/ml) for 24 hrs. The concentrations of NO (A) and PGE2 (B) in the supernatants were measured by Griess and ELISA assay as described in the methods. CG exhibited a significant suppression of LPS-induced NO (A) and PGE2 (B) secretion in concentration-dependent manners. No significant cell death was observed at CG concentrations used in the present study (C). Methyl p-hydroxycinnamate (MH) was used as a reference compound compared with CG. The data are expressed as mean±SD for three (NO) or four (PGE2) independent experiments. *p<0.05 and **p<0.01 indicate statistically significant differences from treatments with LPS alone.

• 

  Fig. 3 CG inhibited LPS-induced iNOS and COX-2 protein expressions in RAW264.7 macrophage cells. (A) RAW264.7 cells were pretreated with indicated concentrations of CG for 1 hr, then incubated with LPS (200 ng/ml) for 24 hrs. The cell lysates were prepared and subjected to Western blotting analysis by using antibodies specific for iNOS and COX-2 as described in the methods. (B) The relative protein levels were quantified by scanning densitometry and normalized to β-actin. The data indicated that CG significantly decreased the LPS-induced overexpression of iNOS and COX-2 in a concentration-dependent manner. The images shown are representatives of three independent experiments that showed consistent results and the relative protein values are expressed as mean±SD for three experiments. *p<0.05 and **p<0.01 indicate statistically significant differences from treatments with LPS alone.

• 

  Fig. 4 CG suppressed the extracellular release of LPS-induced IL-1β and TNF-α cytokines in RAW264.7 macrophage cells. RAW264.7 cells were pretreated with indicated concentrations of CG for 1 hr, then incubated with LPS (200 ng/ml) for 24 hrs. The concentrations of IL-1β (A) and TNF-α (B) in collected cell culture media were measured by ELISA assay as described in the methods. CG meaningfully reduced LPS-stimulated both IL-1β and TNF-α cytokines in a concentration-dependent manner. The values are expressed as mean±SD for three independent experiments. *p<0.05 and **p<0.01 indicate statistically significant differences from treatments with LPS alone.

• 

  Fig. 5 CG significantly attenuated LPS-induced IκBα degradations in RAW264.7 macrophage cells. (A) RAW264.7 cells were pretreated with indicated concentrations of CG for 1 hr, then exposed to LPS (200 ng/ml) for 15 mins. The cell lysates were prepared and subjected to Western blotting analysis by using antibodies specific for IκB as described in the methods. (B) The relative protein levels were quantified by scanning densitometry and normalized to β-actin. The data showed that CG significantly prevented LPS-induced IκB degradations in a concentration-dependent manner. The images shown are representatives of three independent experiments that showed consistent results and the relative protein values are expressed as mean±SD for three experiments. *p<0.05 and **p<0.01 indicate statistically significant differences compared with LPS alone.

• 

  Fig. 6 CG did not affect MAPKs signaling pathways in RAW264.7 macrophage cells. (A) RAW264.7 cells were pretreated with various indicated concentrations of CG for 1 hr, then exposed to LPS (200 ng/ml) for 30 mins. The cell lysates were prepared and subjected to Western blotting analysis by using antibodies specific for total and phosphorylated forms (shown as p-) of JNK, ERK and p38. (B) The relative protein levels of p-JNK, p-ERK and p-p38 were quantified by scanning densitometry and normalized to total JNK, ERK and p38, respectively. The images shown are representatives of three independent experiments that showed consistent results and the relative protein values are expressed as mean±SD for three experiments. The data presented no effect of CG on MAPKs.

• 

  Fig. 7 CG significantly increased Akt phosphorylation in RAW264.7 macrophage cells. (A) RAW264.7 cells were pretreated with various indicated concentrations of CG for 1 hr, then exposed to LPS (200 ng/ml) for another 1 hr. The cell lysates were prepared and subjected to Western blotting analysis by using antibodies specific for total and phosphorylated forms of Akt. (B) The relative protein levels of p-Akt were quantified by scanning densitometry and normalized to total Akt. The data exhibited that CG strongly increased Akt activation in a concentration-dependent manner. The images shown are representatives of three independent experiments that showed consistent results and the relative protein values are expressed as mean±SD for three experiments. *p<0.05, **p<0.01, ***p<0.001 indicate statistically significant differences from treatments with control alone.

• 

  Fig. 8 PI3K/Akt was required for CG-mediated suppression of LPS-induced iNOS and COX-2 expressions and restoration of LPS-induced IκB degradation in RAW264.7 macrophage cells. RAW264.7 cells were pretreated with CG (100 µM) for 1 hr in the presence or absence of wortmannin, then exposed to LPS (200 ng/ml) for 1 hr. The cell lysates were prepared and subjected to Western blotting analysis by using antibodies specific for total and phosphorylated forms of Akt, which showed that wortmannin completely abolished CG-mediated Akt activation (A). The relative protein levels of iNOS and COX-2 were also examined. CG-mediated suppression of LPS-induced iNOS and COX-2 expressions were significantly attenuated with wortmannin but not completely (B). The protein level of IκB was examined. CG-mediated recovery of LPS-induced IκB degradation was significantly abolished with wortmannin (C). The images shown are representatives of three independent experiments that showed consistent results and the relative protein values are expressed as mean±SD for three experiments. *p<0.05, **p<0.01 indicate statistically significant differences from treatments with control alone. #p<0.05 and ##p<0.01 represent statistical significances between indicated groups.

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